Guiding principles: keep the cells as cold as possible at all times; manipulate the cells as gently as possible at all times. The final ligation reaction which combines the cosmid fragments (random products from sonication) and the M13 vector occurs in a very small volume containing µg amounts of vector plus insert product all mixed together. Templates and subsequent sequencing reactions must be derived from individual cosmid fragments. Separation is accomplished by growing plates of E. coli cells, some of which which have been infected with the phage. Electroporation uses strong, brief pulses of electric current to punch holes in the cell membranes of the E. coli. If 1 to 2 µl of the final ligation mix from a library construction is added to the cells just prior to electroporation, then a fraction of the cells will absorb some of the DNA through these holes. Infected cells will replicate the phage and exude single-stand copies of the phage. These cells are combined with a solution of uninfected cells, and this combination is grown overnight on LB plates with IPTG and X-gal. Early in the incubation period, the phage in the infected cells will multiply, and will infect other neighboring cells. M13-infected male E. coli (NM522) are not lysed by the phage. Rather, the single-strand phage are released to infect other nearby cells. M13-infected E. coli grow at (appoximately) one-third of the rate of uninfected cells. Thus, the infected cells make a "plaque" com posed of shallow growth compared to a background lawn of robust growth by the uninfected cells. We are using two different E. coli cells for this procedure. For electroporation we employ DH10B cells (female) because they are robust during electroporation. However, DH10B cells cannot adequately grow M13 phage. Therefore, we add uninfected (male) NM522 cells (after electroporation and before plating) because M13 grows well on NM522 cells (which, however, are fragile during electroporation).
Since M13 vector without yeast DNA insert will give rise to plaques, we use a simple screen to distinguish what we want (M13 with insert) from what we don't want (M13 without insert). The M13 cloning site is within the ß-galactosidase gene. An insert will disrupt the gene. ß-galactosidase is induced by IPTG (in the top agar) and cleaves X-gal (in the top agar). One cleavage product is blue. Therefore, M13 vector will give rise to blue plaques (unwanted). M13 with insert will give rise to clear plaques. Pick clear plaques.
Preparation
1-Check that the cuvette holder has been placed in the freezer. Also check that the settings on the electroporator should be 1.8 V, 25 µFD and 200.
2-Fill ice bucket and place cuvette (0.1 cm-gap cuvettes are used), ligation mix and electrocompetent cells in bucket. (Use either JS5 or DH10B strains of electrocompetent cells)
3-Pipet 1 ml SOC into 1.5-ml screw cap tube.
4-Take up the full ml into pasteur pipet. Place the pipet on the benchtop adjacent to electroporator.
5-Add 1 µl of the ligation mix to 25 µl of electrocompetent cells.
6-Mix twice with pipette and transfer to the cuvette.
7-Cap the cuvette and shake it down once to be sure that the cells are evenly along the bottom of the cuvette.
8-Place the cuvette into the cuvette holder, there is only one orientation that works.
9-Push in the cuvette holder all the way and push both red buttons at the same time, holding them depressed until a beep is heard.
10-Immediately withdraw the cuvette and add the SOC to it, and pipet out the cell mix and pipet into the pre-labeled 1.5-ml screw cap tube.
11-Leave the cells to recover in the 37°C incubator for 10 min.
Plating
1-Mix the top agar as follows per plate:
Top Agar Plate Mixture
2-To ensure that the cells are not cooked alive, be sure that the agar solution is cooled to at least 50°C before adding the cells.
3-Allow plates to cool for 15 min; store the plates inverted at 37°C overnight.
4-Pick clear plaques into 200 µl 15% glycerol in TE.